Immune receptor modifier conjugate and preparation method and use thereof, coupling precursor for preparing same, and compound for synthesizing coupling precursor

ABSTRACT

Disclosed is an immune receptor modifier conjugate, obtained from the reaction between a coupling precursor and a biotic ligand, the coupling precursor being a 9-position aminomethyl benzyl purine biotic coupling precursor, and the biotic ligand being selected from one or more of polypeptide, protein, glycoprotein, polysaccharide, polynucleotide, inactivated cells and inactivated microbes. The immune receptor modifier couplet can be used for immunomodulation, antibody preparation, anti-virus, diabetes, tumor immunomodulation, and tumor bio-immunotherapy. The conjugate compounds or salts thereof can be prepared into various therapeutic drugs, and can be prepared into a compound drug together with other drugs, or pharmaceutically acceptable carrier composites or conjugates. Also disclosed are compounds for synthesizing the coupling precursor and salts thereof.

FIELD OF THE INVENTION

The present invention relates to an immune receptor modifier conjugate,its preparation and use thereof for antitumor, antivirus, diabetes,antibody induction and immunomodulation. In particular, the presentinvention relates to 9-position aminomethyl benzyl purine bioticcoupling precursor for preparing the above-mentioned immune receptormodifier conjugate. In addition, the present invention relates to thecompounds or salts thereof for synthesizing the coupling precursor.

BACKGROUND OF THE INVENTION

Improved immunological effect can often be realized by combinedapplication or conjugation of immune agonists and immune antigens suchas polypeptides, proteins, glycoproteins, polysaccharides,polynucleotides, cell lysates, inactivated cells and inactivatedmicroorganisms (World Chin J Digestol 2005 Sep. 15, 13(17):2078-2081;Vaccine, Volume 23, Issue 45, 1 Nov. 2005, Pages 5263-5270; The J. Clin.Invest. 2011, 121(5), 1782-1796).

The present invention prepares a series of small molecular immuneagonist coupling precursors and conjugates synthesized from such immuneagonist coupling precursors with biotic ligands or immune antigens. Alsodisclosed are the uses of such conjugates for anti-tumor, anti-virus,diabetes, antibody induction and immunomodulation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an immune receptormodifier conjugate with enhanced immunological effect and thepreparation method thereof. The purpose of the present invention is toprovide a coupling precursor for preparing such conjugate and to providecompounds or salts thereof for synthesizing such coupling precursor. Afurther purpose of the present invention is to provide the use of suchimmune receptor modifier conjugate in immunomodulation, antibodypreparation, anti-virus, diabetes, tumor immunomodulation and tumorbiological-immunotherapy.

According to one aspect of the present invention, provided is an immunereceptor modifier conjugate which is obtained by reacting a couplingprecursor with a biotic ligand, wherein the conjugate is a compound ofgeneral formula (I):

wherein R² represents the biotic ligand, X¹ represents OH or SH, R¹represents linear alkyl, branched alkyl, substituted alkyl,unsubstituted alkyl or alkoxyalkyl, X² represents a coupling group; mand n each are an integer selected from 1 to 10, m is the number ofsmall molecular agonist (m is defined as coupling degree), and n is thenumber of the biotic ligand.

When the coupling precursor is a compound of formula 1:

X² represents the group thiocarbonyl

When the coupling precursor is a compound of formula 2:

X² represents the group

When the coupling precursor is a compound of formula 3:

X² represents the group

in which u is an integers selected from 0 to 12;

and

When the coupling precursor is a compound of formula (4):

X² represents the group

in which PEG represents a polyethylene glycol group.

Among the above-mentioned immune receptor modifier conjugates, thebiotic ligand is one or more selected from polypeptide, protein,glycoprotein, polysaccharide, polynucleotide, inactivated cells andinactivated microorganisms.

Among the above mentioned immune receptor modifier conjugates, PEG is apolyethylene glycol group

such as a di-polyethylene glycol group

a tri-polyethylene glycol group

and a tetra-polyethylene glycol group

When the coupling precursor is a compound of formula 3 or 4, by usingthe biotic ligand R², such as OCT4, SOX2, NANOG, MUC1, MG7, POSTN,Twist, Anxa1, Akt, CD47, Sp17, PSMA, M2e (monomer and tetramer),NP₃₆₆₋₃₇₄ (9-peptide epitope), FOX01, PEAK1, HER2, MMP-10, PD-L1, PD-1,and SGLT2, the typical representative structures of the proteinconjugates formed are as follows:

In formulae (5) and (6), the expression “or R²” means that OCT4 and SOX2can be substituted by other antigens, proteins or polypeptides, whereinthe substitution by NANOG results in compounds 5-3 and 6-3 respectively;the substitution by MUC1 results in compounds 5-4 and 6-4 respectively;the substitution by MG7 results in compounds 5-5 and 6-5 respectively;the substitution by POSTN results in compounds 5-6 and 6-6 respectively;the substitution by Twist results in compounds 5-7 and 6-7 respectively;the substitution by Anxa1 results in compounds 5-8 and 6-8 respectively;the substitution by Akt results in compounds 5-9 and 6-9 respectively;the substitution by CD47 results in compounds 5-10 and 6-10respectively; the substitution by Sp17 results in compounds 5-11 and6-11 respectively; the substitution by PSMA results in compounds 5-12and 6-12 respectively; the substitution by M2e (monomer and tetramer)results in compounds 5-13 and 6-13 respectively; the substitution byNP₃₆₆₋₃₇₄ (9-peptide epitope) results in compounds 5-14 and 6-14respectively; the substitution by SGLT2 results in compounds 5-15 and6-15 respectively; the substitution by PEAK1 results in compounds 5-16and 6-16 respectively; the substitution by HER2 results in compounds5-17 and 6-17 respectively; the substitution by MMP-10 results incompounds 5-18 and 6-18 respectively; the substitution by PD-L1 resultsin compounds 5-19 and 6-19 respectively; and the substitution by PD-1results in compounds 5-20 and 6-20 respectively.

The aforementioned compounds are shown in tables 1 and 2 below:

TABLE 1 Conjugates formed by the coupling precursor of formula 3 andvarious biotic ligands MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 AktCD47 Sp17 Conjugate 5-3 5-4 5-5 5-6 5-7 5-8 5-9 5-10 5-11 R² PSMA M2eNP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 5-12 5-13 5-145-15 5-16 5-17 5-18 5-19 5-20

TABLE 2 Conjugates formed by the coupling precursor of formula (4) andvarious biotic ligands MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 AktCD47 Sp17 Conjugate 6-3 6-4 6-5 6-6 6-7 6-8 6-9 6-10 6-11 R² PSMA M2eNP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 6-12 6-13 6-146-15 6-16 6-17 6-18 6-19 6-20

Among them, OCT4, SOX2 and NANOG are tumor stem cell antigen proteins(Chinese Medicine Herald, 2011, 8(8):17-20; Chinese Bulletin of LifeSciences, 2004, 16(3):129-134); MG7 is a stomach cancer-related antigenprotein (China Oncology, 2010, 20(4): 312-313); MUC1 is an antigenrelated to various tumors (European Journal of Organic Chemistry, 2011,20(21): 3685-3689); POSTN is a tumor-related protein (Nature, 2012, 481:85-89); Twist and Akt are both tumor-related antigens (Journal ofShenyang Medical College, 2010, 12(3): 182-184); Anxa1 is specificallyexpressed in various tumor tissues (PNAS, 2011, Oct. 3, 19587-19592;Oncology Progress, 2010, Jan. 8(1):63-66); CD47 is highly expressed onthe surface of almost every type of cancer cells (Science: TranslationalMedicine, 22 Dec. 2010, Vol 2, Issue 63 63ra94); Sp17 is a tumor antigenhaving very high expression in ovarian carcinoma cells (PloS ONE, 2010,5(5), e10471, 1-13); PSMA is a special biomarker of prostate cancer(Cancer, 1998, 82(11): 2256-2261); M2e and NP₃₆₆₋₃₇₄ are epitopepolypeptides in the conserved protein of influenza virus A; SGLT2 is afunctional protein related to sugar metabolism (Chinese Journal ofMedicinal Chemistry, 2011, Vol. 21, No. 4, p 322); PEAK1 is a biomarkerof early pancreatic cancer (Cancer Res. 2012 May 15; 72(10):2554-64);HER2 is a proto-oncogene human epidermal growth factor receptor 2(MEDICAL JOURNAL OF CASC, 2002, 4 (1), 69-70); MMP-10 is a proteinclosely related to the occurrence and metastasis of lung carcinoma(Anticancer Res. 2007 July-August; 27(4C): 2791-5); and PD-L1 and PD-1are a ligand and a receptor of tumor immune escape, respectively (N.Engl. J. Med., 2012, Jun. 2, 1-11).

When the coupling precursor is a compound of formula 1 or 2, typicalrepresentative synthesis and structure formulae of the polypeptideconjugates formed from the coupling precursor and biotic ligand R² (suchas MUC1

MG7 and M2e (monomer)) are as follows:

For the immune receptor modifier conjugates of this invention, suitablebiotic ligands are not limited to the polypeptides or proteins as listedabove, and can also be the tumor antigens as shown is table 3.

TABLE 3 Suitable biotic ligands in the form of tumor antigens Serial No.1 2 3 4 5 6 7 8 9 Antigen WT1 MUC2 LMP2 HPV E6 EGFR_(v)III HER-2/neuIdiotype MAGE A3 p53 monmutant E7 Percentage of positive 0.37 1.0 0.370.23 0.37 0.37 1.0 0.37 0.37 expression and cells (0.07) Stem cellperformance (0.04) 1.0 1.0 1.0 0.73 1.0 0.66 0.66 1.0 1.0 Number ofparents with 1.0 1.0 1.0 0.16 0.11 0.11 0.14 1.0 1.0 cancer cell antigen(0.04) Amount of antigen (0.04) 1.0 1.0 1.0 1.0 0.13 1.0 1.0 1.0 1.0Constitutive expression in 0.95 0.25 0.95 0.95 1.0 0.25 1.0 0.95 0.95cells (0.02) Serial No. 10 11 12 13 14 15 16 17 18 Antigen NY-ESO-1 PSMAGD2 CEA MelanA/V Ras Gp100 P53 mutant Proteinase3 MART1 Mutant (PR1)Percentage of positive 0.37 1.0 1.0 0.37 0.37 0.23 0.37 1.0 0.37expression and cells (0.07) Stem cell performance (0.04) 1.0 0.2 0.20.66 0.2 1.0 0.2 0.77 0.2 Number of parents with 0.11 1.0 1.0 1.0 1.00.16 1.0 0.14 1.0 cancer cell antigen (0.04) Amount of antigen (0.04)1.0 1.0 1.0 1.0 1.0 0.13 1.0 0.13 0.13 Constitutive expression in 0.951.0 0.62 0.25 0.95 0.95 0.95 0.95 0.95 cells (0.02) Serial No. 19 20 2122 23 24 25 26 27 Antigen Bct-abo Tyrosinase Survivin PSA hTERT SarcocnaEphA2 PAP ML-IAP translocation breakpoints Percentage of positive 0.230.37 0.37 0.08 0.23 1.0 0.37 0.23 0.37 expression and cells (0.07) Stemcell performance (0.04) 1.0 0.2 0.66 0.66 1.0 1.0 0.2 0.2 0.2 Number ofparents with 0.16 1.0 1.0 0.16 0.16 1.0 1.0 0.16 1.0 cancer cell antigen(0.04) Amount of antigen (0.04) 0.13 1.0 1.0 1.0 1.0 0.13 1.0 1.0 1.0Constitutive expression in 0.95 0.95 0.95 0.95 0.25 0.95 0.95 1.0 0.25cells (0.02) Serial No. 28 29 30 31 32 33 34 35 36 Antigen AFP EpCAMERG(TMR NA17 PAX3 ALK Androgen Cyclin B1 Polysialic PSS2 ETS receptorfusion gene) Percentage of positive 0.37 1.0 0.37 0.00 0.08 1.0 0.370.32 1.0 expression and cells (0.07) Stem cell performance (0.04) 1.01.0 0.66 0.00 0.2 1.0 0.66 0.66 0.2 Number of parents with 1.0 1.0 1.01.0 1.0 1.0 1.0 1.0 1.0 cancer cell antigen (0.04) Amount of antigen(0.04) 1.0 1.0 0.13 0.13 1.0 0.27 1.0 1.0 1.0 Constitutive expression in0.25 1.0 0.95 0.95 0.95 0.95 0.95 0.95 1.0 cells (0.02) Serial No. 37 3839 40 41 42 43 44 45 Antigen MYCN Rhoc TRP-2 GD3 Fucosyl Medothelin PSCAMEGE A1 sLe(a) GM1 Percentage of positive 0.37 0.37 0.37 1.0 1.0 0.370.37 0.00 1.0 expression and cells (0.07) Stem cell performance (0.04)0.2 0.66 1.0 0.2 0.2 0.66 0.2 0.2 0.2 Number of parents with 1.0 1.0 1.01.0 1.0 1.0 0.16 0.16 1.0 cancer cell antigen (0.04) Amount of antigen(0.04) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Constitutive expression in0.95 0.95 0.95 1.0 1.0 1.0 1.0 0.95 0.25 cells (0.02) Serial No. 46 4748 49 50 51 52 53 54 Antigen CYP1B1 PLAC1 GM3 BORIS Tn GloboH ETV6-ANY-BR-1 RGS5 ML Percentage of positive 1.0 0.37 0.37 0.08 0.37 0.37 0.370.36 0.35 expression and cells (0.07) Stem cell performance (0.04) 0.20.2 0.2 0.66 0.2 0.00 0.00 0.00 0.00 Number of parents with 1.0 0.11 1.00.16 1.0 1.0 0.00 0.39 1.0 cancer cell antigen (0.04) Amount of antigen(0.04) 1.0 1.0 1.0 1.0 1.0 0.12 1.0 0.12 0.00 Constitutive expression in0.95 1.0 0.25 0.95 1.0 1.35 1.0 1.0 0.35 cells (0.02) No. 55 56 57 58 5960 61 62 63 Antigen SART3 STn Carbonic PAX5 OY-TES1 Sperm protein LCKHMWMAA AKAP-4 anhydrase 17 IX Percentage of positive 0.35 0.34 0.34 0.330.32 0.30 0.28 0.27 0.26 expression and cells (0.07) Stem cellperformance (0.04) 0.00 0.00 0.00 0.00 0.00 0.1 0.00 0.1 0.1 Number ofparents with 1.0 1.0 1.0 0.39 0.1 0.11 1.0 0.11 0.11 cancer cell antigen(0.04) Amount of antigen (0.04) 0.00 0.25 0.00 1.0 1.0 0.25 0.00 0.000.12 Constitutive expression in 0.35 0.23 0.35 0.21 0.54 0.54 0.35 0.350.54 cells (0.02) No. 64 65 66 67 68 69 70 71 72 Antigen SSX2 XAGE 1B7H3 Legumain Tie 2 Page4 VEGFR2 MAD-CT-1 FAP Percentage of positive0.26 0.23 0.22 0.19 0.18 0.17 0.16 0.15 0.14 expression and cells (0.07)Stem cell performance (0.04) 0.00 0.00 0.00 0.1 0.1 0.00 0.1 0.00 0.1Number of parents with 0.39 0.1 0.00 0.11 0.11 0.00 0.11 0.1 0.00 cancercell antigen (0.04) Amount of antigen (0.04) 0.25 0.00 0.25 0.00 0.000.12 0.12 0.00 0.00 Constitutive expression in 0.54 0.54 0.35 0.35 0.230.21 0.1 0.54 0.1 cells (0.02) Serial No. 73 74 75 Antigen PDGFR-bMAD-CT-2 Fos-related antigen 1 Percentage of positive 0.14 0.14 0.13expression and cells (0.07) Stem cell performance (0.04) 0.0 0.0 0.1Number of parents with 0.11 0.1 0.11 cancer cell antigen (0.04) Amountof antigen (0.04) 0.12 0.00 0.00 Constitutive expression in 0.1 0.54 0.1cells (0.02) (Characteristic, preparation and source of the tumorantigens as shown in table 2 may be found in Clin Cancer Res, 2009;15(17), 5323-5337).

Preparation of the Coupling Precursor:

Synthesis of Coupling Precursor 1—Synthesis Protocol:

Method for preparing compound of formula 7 is described in WO 2009005687for compound 1096159-02-2P.

Synthesis Process: Compound 7 (5 g) was dissolved into methanol (100 ml)and active nickel (0.5 g) was added. The mixture was reduced byhydrogenation at room temperature under 3 atm. for 24 h. After thecatalyst nickel was filtered off, the residue was concentrated underreduced pressure to small volume, frozen at −10° C. for 12 h, andprecipitated to give a solid product 8 (3 g, yield 60%). Melting point:265-267° C. MS (ESI, M+1): 345.

Compound 8 (1 g, 2.9 mmol) was mixed with carbon disulfide (2 ml),triethylamine (0.5 ml), DMAP (0.1 g), (BOC)₂O (640 mg) anddichloromethane (50 ml). The mixture was stirred at room temperature for2 h, heated under reflux for 4 h, concentrated under reduced pressure,and separated by silica gel column chromatography (5%methanol-dichloromethane) to give compound 1 (0.7 g, yield 65%). Meltingpoint: 221-223° C., MS (ESI, M+1): 373.

Synthesis of Coupling Precursor 2—Synthesis Protocol:

Synthesis Process: Compound 8 (1 g) was mixed with compound 9 (0.78 g)in DMF (30 ml), stirred under room temperature for 2 h, concentratedunder reduced pressure, and separated by silica gel columnchromatography (ethyl acetate) to give compound 2 (1.1 g, yield 82%).Melting point 203-205° C. MS (ESI, M+1): 496.

Synthesis of Coupling Precursor 3-1 (u=2)—Synthesis Protocol:

Synthesis Process: Compound 8 (1 g) was mixed with succinic anhydride(0.3 g) in DMF (50 ml) and stirred at room temperature for 12 h.N-hydroxy succinimide (0.34 g) and equal molar DCC were added. Themixture was stirred at room temperature for another 12 h to give asolution of compound 3 in DMF. The solution was distilled under reducedpressure to dryness. The resultant solid residue was extracted withethyl acetate (50 ml), and then equal volume of diethyl ether was added.The mixture was frozen at −10° C. for 12 h and precipitated to give asolid 3 (u=2) (0.6 g, yield 38%). MS (ESI, M+1): 542.

Synthesis of Coupling Precursor 4-1—Synthesis Protocol

The reference for synthesis of compound 11: PCT patent application No.2011134669, published on Nov. 3, 2011.

Note: 1) regioselective, scale up; 2) regioselective, scale up; 3) scaleup, reactants: 3, reagents: 3, solvents: 3, steps: 3, stages: 3.

Synthesis of compound 12: the synthesis protocol is analogous to thesynthesis of compound 8, yield: 88%, MS (ESI, M+1): 329.

Synthesis of compound 13: Compound 12 (1 g) was dissolved intodichloromethane (50 ml). Then bromine (10 ml) was added and the mixturewas stirred at room temperature for 12 h. Air was blown through themixture such that the excessive bromine was removed and absorbed intosaturated sodium bicarbonate solution. The solid residue was filtered togive a pale yellow solid 13 (1 g), with a yield of 85% and mass spectra(ESI, M+1): 407.

Synthesis of compound 15: Compound 13 (0.5 g) was dissolved intoanhydrous DMF (10 ml) and compound 14 (0.24 g) was added. The mixturewas stirred homogenously, and then added with DCC (0.26 g) at 0° C. Themixture was stirred at 0° C. for 2 h and then at room temperature foranother 12 h. DCU was filtered off and the remaining solution wasdistilled under reduced pressure to dryness. To the residue was added 50ml of saturated sodium bicarbonate solution. The mixture was stirred at40° C. for 4 h. Under cooling, the mixture was adjusted to pH 4 withconcentrated hydrochloric acid. The precipitated solid was filtered,washed with water, and dried to give compound 15 (0.5 g, yield 72%). MS(ESI, M+1):567.

Synthesis of compound 16: Compound 15 (0.4 g) was dissolved intomethanol (20 ml), and thiourea (1 g) was added. The mixture was heatedunder reflux for 12 h, cooled and filtered. The filtrate wasconcentrated under reduced pressure and the residue was dissolved into5% sodium carbonate solution (10 ml). Under cooling, the mixture wasadjusted to pH 4 with concentrated hydrochloric acid. The precipitatedsolid was filtered, washed with water and dried to give compound 16(0.23 g, yield 65%). MS (ESI, M+1):521.

Synthesis of coupling precursor 4: Compound 16 (0.15 g) was dissolvedinto anhydrous DMF (5 ml), and N-hydroxy succinimide (0.04 g) was added.The mixture was stirred homogenously and then added with DCC (0.06 g) at0° C. The resultant mixture was stirred at 0° C. for 2 h and then atroom temperature for another 12 h. DCU was filtered off and the filtratewas distilled under reduced pressure to dryness. The remaining solid wasextracted with ethyl acetate (10 ml). To the extraction was added equalvolume of diethyl ether. The mixture was frozen at −10° C. for 12 h andprecipitated to give a solid 4 (0.05 g, yield 32%). MS (ESI, M+1):618.

For immune therapy and immunomodulation of malignant tumor, the immunereceptor modifier conjugate may be administered by intraperitoneal,subcutaneous, intramuscular or intravenous injection. Alternatively, theimmune receptor modifier conjugate may be administered by in vivore-transfusing the isolated immune cells after co-culturing the immunecells (for example, dendritic cells, natural killer (NK) cells,lymphocyte, monocyte/macrophage, granulocyte, etc.) with the immunereceptor modifier conjugate of this invention.

The immune receptor modifier conjugate of the present invention can beused for immunomodulation, antibody preparation, anti-virus, diabetes,tumor immunomodulation and tumor biological-immunotherapy. Theabove-mentioned conjugates or salts thereof can be formulated intotherapeutic drugs suitable for such therapies, or formulated intocompound drugs with other pharmaceuticals, or formulated into complexesor combinations with pharmaceutically acceptable carriers. The presentimmune receptor modifier conjugates or salts thereof can be present atvarious ratios in the therapeutic preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by the following figuresand specific examples.

In the figures:

FIG. 1 is a graph showing the comparison of IFN-γ induction by conjugate5-4, in which T7 is coupling precursor 1 and T7-MUC1 is conjugate 1-2.

FIG. 2 is a graph showing the comparison of IL-12 induction by conjugate2-1, in which T7 is coupling precursor 2 and T7-M2e is conjugate 2-1.

FIG. 3 is a diagram schematically showing the tumor-inhibiting effectsof conjugate 5-4 (m=1), in which T7 is coupling precursor 10 and T7-MUC1is conjugate 5-4 (m=1), *p<0.001.

FIG. 4 is a diagram schematically showing the tumor-inhibiting effectsof conjugate 5-4 (m=1), in which T7 is coupling precursor 10 and T7-MUC1is conjugate 5-4 (m=1), *p<0.001.

FIG. 5 is a diagram schematically showing the in vivo antibody-inducingeffect of the influenza virus conserved protein M2e conjugate (T7-M2e),compared with original antigen M2e.

FIG. 6 is a diagram schematically showing the immune-induction effect ofurine glucose reabsorption functional protein SGLT2, in which T7-SGLT2is conjugate 5-15.

DETAILED DESCRIPTION OF THE INVENTION

Source of the antigens: the molecular weight of all proteins may befound in the international protein database(http://www.uniprot.org/uniprot/P48432).

Source of proteins or polypeptides: Except the proteins or polypeptidesfor which the specific preparation or synthesis methods have beendescribed, other polypeptides are synthesized by Hybio PharmaceuticalCo., Ltd.

Proteins are purchased from or synthesized by the following companies:

GenScript Inc., Nanjing, China (http://www.genscript.com.cn/index.html);

Novoprotein Scientific Inc., Shanghai, China (http://www.sinobio.net/);

Sino Biological Inc., Beijing, China (http://www.sinobiological.cn/);

Abnova Corporation (http://www.abnova.com/cn/).

Mass spectrometer for material identification: LDI-1700 MALDI-TOF massspectroscopy (Linear Scientific Inc., USA).

The method for determining coupling degree by mass-spectrometry is, forexample, as follows:

The average molecular weight of POSTN is 86 kDa (see, World JGastroenterol, 2007 Oct. 21; 13(39); 5261-5266;http://www.sinobiological.cn/Periostin-Protein-g-465.html). Themolecular weight of the resultant coupling product 5-6 is 88132, asdetermined by mass spectrum. The coupling degree is calculated asfollows: (88132-86000)/427=4.99≈5 (the molecular weight 427 is obtainedby subtracting one H₂O molecular from molecular weight of compound 3).Thus, it can be determined that 5 monomers of compound 3 were coupled(coupling degree m=5).

The preparation of the present coupling conjugate is further illustratedby the following examples.

Synthesis of Compound 1-1:

MG7 (0.37 mmol) and compound 1 (0.88 mmol) were mixed and dissolved intoabsolute methanol, and then anhydrous triethylamine (1.12 mmol) wasadded. The mixture was reacted at 45° C. for 4 h. The solvent wasremoved by evaporating under reduced pressure and the residue wasseparated by silica gel column chromatography (10%methanol-dichloromethane) to give compound 1-1 (55 mg, yield 23%). MS(ESI): theoretical m/z 969.1516, found 970.1518 (M+H).

Synthesis of Compound 1-2:

Analogous to the synthesis of compound 1-1, MUC1 (epitope) was mixedwith compound 1 at a molar ratio of 1:1, and then 1.5-fold molar oftriethylamine was added. The resultant mixture was allowed to react inmethanol at 20° C. for 12 h. For work up, please refer to “Synthesis ofcompound 1-1”. This gave compound 1-2 (yield 20%). MS (ESI): theoreticalm/z 3341.72, found 3342.75 (M+H).

Synthesis of Compound 1-3:

Analogous to the synthesis of compound 1-1, M2e was mixed with compound1 at a molar ratio of 1:1, and then 1.5-fold molar of triethylamine wasadded. The resultant mixture was allowed to in methanol at 20° C. for 12h. For work up, please refer to “Synthesis of compound 1-1”. This gavecompound 1-3 (yield 25%). MS (ESI): theoretical m/z 3303.32, found3304.33 (M+H).

Synthesis of Compound 2-1:

Compound 2 (0.22 mmol) was mixed with M2e (0.1 mmol) in anhydrous DMSO(10 ml). The mixture was stirred at room temperature for 12 h. To themixture H₂O (100 ml) was added. The resultant mixture was lyophilized toremove solvents and the residue was separated by silica gel columnchromatography (10% methanol-dichloromethane) to give compound 2-1 (56mg, yield 15%). MS (ESI): theoretical m/z 3761.71, found 3761.75 (M+H).

Synthesis of Compound 5:

1) The expression and preparation of OCT4 are described in Progress inModern Biomedicine, Vol. 10, NO. 9, May, 2010, 1610-1612).

2) Synthesis of Compound 5 (m=5):

OCT4 (10 mg, average molecular weight: 38216) was dissolved into PBSsolution (10 ml). A solution of compound 3 (u=2, 50 mg) in 2 ml DMSO wasmixed with equimolar NHS, and then equimolar EDC was added. The mixturewas stirred at room temperature for 2 h. Subsequently, the solution ofOCT 4 in PBS was added and the resultant mixture was stirred overnightat 10° C. The mixture was separated with PD-10 desalting column(Amersham disposable PD-1 desalting column). The eluates containingconjugate 5 were combined (detected by the absorptance at 320 nm) andlyophilized. The average molecular weight was 40348, as determined byMS. Thus, it was determined that the product 5 comprises five monomersof compound 3 (u=2), i.e. coupling degree m=5. Compound 5 (m=1, 2, 3, 4)was synthesized with compound 3 (u=2) at different molar ratios by thesame method.

Synthesis of Compound 5-4:

1) Methods for preparing MUC1 (epitope) may be found in the references,such as, Proc Natl Acad Sci. 2011, 109(1): 261-266; Angew Chem Int EdEngl, 2010, 49(21): 3688-3692; Angew Chem Int Ed Engl, 2011, 50(7):1635-1639; European Journal of Organic Chemistry, 2011, 20(21):3685-3689; and Chemistry, 2011, 17(23): 6396-6406. According to compound11 shown in the reference Proc Natl Acad Sci. 2011, 109(1): 261-266, themolecular weight of MUC1 (epitope) is 2967.

2) Coupling precursor 3 (u=2, 10 mg, 0.019 mmol) was dissolved into DMSO(2 ml) and MUC1 (epitope) (59 mg, 0.02 mmol) was added. The mixture wasstirred at room temperature for 2 h, and then H₂O (20 ml) was added. Thesolvent was removed by lyophilization and the residue was separated bysilica gel column chromatography (10% methanol-dichloromethane) to givecompound 5-4 (16 mg, yield 25%). MS (ESI): 3396.6 (M+H). Coupling degreem=1.

Synthesis of Compound 5-14:

Analogous to the synthesis of compound 5-4, except that MUC1 (epitope)was replaced by NP₃₆₆₋₃₇₄ (ASNENMDAM), other solvents and reactants areused in identical molar ratios to give compound 5-14, MS (ESI): 1424.54(M+H). Coupling degree m=1.

Synthesis of the Following Conjugates:

If the antigen is a protein, the synthesis is analogous to the synthesisof compound 5. If the antigen is a polypeptide (with the number of aminoacids being less than 50), then follow the synthesis method of compound5-4 (the solvents and reactants were in identical molar ratios, and thedetermination of reaction time, temperature, molecular weight andcoupling degree, as well as the steps and conditions of the method werealso identical). The conjugates obtained are shown in the table below.

Wherein R² represents polypeptide or protein

MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate5-3 5-4 5-5 5-6 5-7 5-8 5-9 5-10 5-11 Molecular 36326 3396 1272 8813222233 40420 50724 36067 18259 weight Coupling 4 1 1 5 3 4 6 2 2 degreeR² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 5-125-13 5-14 5-15 5-16 5-17 5-18 5-19 5-20 Molecular 86890 3357 1424 75029194385 140469 55430 35834 33353 weight Coupling 6 1 1 5 3 6 3 6 4 degree

Preparation of Conjungate 6:

1) SOX2 is prepared according to Journal of Huazhong Normal University(Nat. Sci.), 2008, 42 (1), 102-105. Sp17 is prepared according toChinese Journal of Pathophysiology, 2001, 17(10), 1019-1021. PSMA isprepared according to The Journal of Biomedical Research (NaturalScience), 2010, 30(11): 1608-1611.

2) Synthesis of Compound 6 (m=3):

SOX2 (10 mg, average molecular weight: 34310) was dissolved into PBSsolution (10 ml). A solution of compound 4 (50 mg) (PEG=ethylene glycolgroup) in DMSO (2 ml) was mixed with equimolar NHS, and then equimolarEDC was added. The mixture was stirred at room temperature for 2 h.Subsequently, the solution of SOX2 in PBS was added and the resultantmixture was stirred overnight at 10° C. The mixture was separated onPD-10 desalting column (Amersham disposable PD-1 desalting column). Theeluates containing compound 6 (detected by the absorptance at 320 nm)were combined and lyophilized. The average molecular weight was 35817,as determined by MS. Thus, it was determined that the product 6comprises three monomers of compound 4 (PEG=ethylene glycol group), i.e.coupling degree m=3. Compound 6 (m=1, 2, 4) was synthesized withcompound 4 (PEG=ethylene glycol group) used at different molar ratios bythe same method.

Synthesis of Compound 6-3 (m=4):

Synthesis of compound 6-3 (m=4) was analogous to the synthesis ofcompound 6. The molecular weight of NANOG was 34620. Compound 6-3 (m=4)was analyzed to have a molecular weight of 36630.24. Compound 6-3 (m=1,2, 3, 5) was synthesized with compound 4 (PEG=ethylene glycol group) atdifferent molar ratios by the same method.

Synthesis of Compound 6-14 (the Ligand in General Formula I isNP₃₆₆₋₃₇₄, n=3):

Compound 4 (PEG=ethylene glycol group) (11 mg, 0.02 mmol) was dissolvedinto DMSO (2 ml), and then NP₃₆₆₋₃₇₄ trimer (59.1 mg, 0.02mmol) wasadded. The mixture was stirred at room temperature for 2 h. To themixture with 20 ml water was added. The resultant mixture waslyophilized to remove solvents, and then the residue was separated bysilica gel column chromatography (10% methanol-dichloromethane) to givecompound 6-14 (in which R² is NP₃₆₆₋₃₇₄ trimer) (17 mg, yield 25%). MS(ESI): 3460.8 (M+H). Coupling degree m=1.

Synthesis of the Following Conjugates:

If the antigen is a protein, synthesis is analogous to the synthesis ofcompound 6. If the antigen is a polypeptide (with the number of aminoacids being less than 50), then follow the synthesis process of compound6-14 (the solvents and reactants were in identical molar ratios, and thedetermination of reaction time, temperature, molecular weight andcoupling degree, as well as the steps and conditions of the method werealso identical). The conjugates obtained are shown in the table below.

R² represents polypeptide or protein

MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate6-3 6-4 6-5 6-6 6-7 6-8 6-9 6-10 6-11 Molecular 36630 3469 2257 8851222461 40724 51362 36219 18411 weight Coupling 4 1 1 5 3 4 6 2 2 degreeR² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 6-126-13 6-14 6-15 6-16 6-17 6-18 6-19 6-20 Molecular 87346 3433 3460 75410194614 140925 55659 36290 33657 weight Coupling 6 1 1 5 3 6 3 6 4 degree(Note: R² of conjugate 6-14 is NP₃₆₆₋₃₇₄ trimer; and R² of conjugate 6-5is MG7 trimer)

Method for Assaying Bioactivity

1. Method for Assaying Immune Factors:

Method: ELISA

Reagents and Conditions:

Human peripheral blood mononuclear cells were separated bycentrifugation sedimentation. The centrifugator was Ficoll-Hypaque. Theseparated cells were suspended in RPMI1640 medium, and then 10% of FBS,L-glutamine, and penicillin/streptomycin (RP10, Invitrogen) were added.The resultant mixture was placed into 96-well plate. The cells werestimulated by the compounds of the present invention at a concentrationof 0.1-10 μM, and cultured at 37° C. with 5% CO₂ for 24 h. The levels ofγ-interferon and interleukin-12 were measured using Luminex (Austin,Tex.), FIGS. 1 and 2 showed the in vitro immunity-induction effects ofconjugate 5-4 and conjugate 2-1, respectively. The results shown in thefigures were average of three separate experiments.

2. Method for Assaying Anti-Tumor Effect:

The method for assaying in vivo anti-tumor effect by the conjugates ofthis application such as conjugate 5-4 in mice was as follows:

Cell: 4T1, mouse breast carcinoma cells, 1×10⁶ cells/mouse.

Animals: 5-week-old BALB/C mice (the number of male mice is identicalwith female mice).

Tumor transplantation: Tumor cells were injected subcutaneously onleft/right sides of a mouse.

Administration: Intraperitoneal injection (0.125 mg/mouse/injection, PBSsolution). Drugs were administered three times in total: one week beforetransplantation, on the day of transplantation and 7 days aftertransplantation.

The health state and tumor size of the mice were monitored (via visualobservation). After 14 days, the mice were killed. Blood samples werecollected from each mouse for testing cytokines, and the tumors weretaken out to measure the weight, length and width so as to calculatetheir volumes. The method used for testing in vivo anti-tumor effect by,for example, compound 6-17, in mice was the same as those describedabove, except that the cells used were mouse lung carcinoma cells. FIGS.3 and 4 showed the tumor-inhibiting effects. FIG. 5 showed the antibodyinduction effect. FIG. 6 showed immunity-induction effect ofdiabetes-related proteins.

3. Antibody Induction Method (Taking Compounds 1-3 and 2-1 as theExamples):

36 female BALB/c mice (5 to 6-week-old, about 16 g) were randomlyassigned into six cages, 6 mice each. The mice were provided withstandard diet prepared by animal center and cooled boiled water on dailybasis. After purchase, the mice were fed for one week to adapt theenvironment. After health examination, the experiments were started. Theexperimental animals were randomly divided into six groups, as shown inTable 4:

TABLE 4 Grouping and treatment of experimental animals Group Immune dose(per mouse) PBS group 200 μL sterile PBS TLR-7 agonist group 18 μM TLR-7agonist M2e group 18 μM M2e antigen TLR-7 and M2e mixture group 200 μLmixture (18 μM TLR-7 and M2e, mixed at a ratio 1:1 by volume) Conjugate(TLR-7:M2e = 1:1) group 18 μM conjugate (TLR-7:M2e = 1:1) (conjugate1-3) Conjugate (TLR-7:M2e = 2:1) group 18 μM conjugate (TLR-7:M2e = 2:1)(conjugate 2-1)

200 μl of the drugs were administered intraperitoneally to the abovegroups of mice, respectively. Booster immunization was performed withthe same antigen and dosage on day 14 after initial immunization. Bloodsamples were collected by cutting rat tail for three times: beforeinitial immunization, before booster immunization and 7 days afterbooster immunization. The blood samples were placed into 1.5 mlEppendorf tubes at 4° C. for several hours, and then centrifuged at 3000rpm for 15 min to separate serum. M2e-specific antibodies in the immuneserum were assayed using standard ELISA method.

1. An immune receptor modifier conjugate, characterized in that the conjugate is obtained by the reaction between a coupling precursor and a biotic ligand, and is a compound of formula (I):

wherein R² represents the biotic ligand, X¹ represents OH or SH, R¹ represents linear alkyl, branched alkyl, substituted alkyl, unsubstituted alkyl or alkoxyalkyl, X² represents a coupling group; and m and n each are an integer selected from 1 to 10; When the coupling precursor is a compound of formula 1:

X² represents the group thiocarbonyl

When the coupling precursor is a compound of formula 2:

X² represents the group

When the coupling precursor is a compound of formula 3:

X² represents the group

in which u is an integer selected from 0 to 12; or when the coupling precursor is a compound of formula 4:

X² represents the group

in which PEG represents a polyethylene glycol group.
 2. The immune receptor modifier conjugate of claim 1, characterized in that the biotic ligand is one or more selected from the group consisting of polypeptide, protein, glycoprotein, polysaccharide, polynucleotide, inactivated cells and inactivated microorganisms.
 3. The immune receptor modifier conjugate of claim 1, characterized in that the conjugate comprises the compounds of formulae 1-1, 1-2, 1-3 and 2-1:


4. The immune receptor modifier conjugate of claim 1, characterized in that the conjugate comprises:

wherein the biotic ligand R² represents polypeptide or protein; MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate 5-3 5-4 5-5 5-6 5-7 5-8 5-9 5-10 5-11 Molecular 36326 3396 1272 88132 22233 40420 50724 36067 18259 weight Coupling 4 1 1 5 3 4 6 2 2 degree R² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 5-12 5-13 5-14 5-15 5-16 5-17 5-18 5-19 5-20 Molecular 86890 3357 1424 75029 194385 140469 55430 35834 33353 weight Coupling 6 1 1 5 3 6 3 6 4 degree


5. The immune receptor modifier conjugate of claim 1, characterized in that the conjugate comprises:

wherein the biotic ligand R² represents polypeptide or protein; MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate 6-3 6-4 6-5 6-6 6-7 6-8 6-9 6-10 6-11 Molecular 36630 3469 2257 88512 22461 40724 51362 36219 18411 weight Coupling 4 1 1 5 3 4 6 2 2 degree R² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 6-12 6-13 6-14 6-15 6-16 6-17 6-18 6-19 6-20 Molecular 87346 3433 3460 75410 194614 140925 55659 36290 33657 weight

wherein R² in conjugate 6-14 is NP₃₆₆₋₃₇₄ trimer, and R² in conjugate 6-5 is MG7 trimer.
 6. A coupling precursor for preparing the immune receptor modifier conjugate of claim 1, characterized in that the coupling precursor comprises compounds of formulae 1, 2, 3 and 4:

wherein u is an integer selected from 0 to 12; and


7. A compound for synthesizing the coupling precursor of claim 6, characterized in that the compound is a compound of formula 10, 12, 13, 15 or 16:

or the salts thereof.
 8. A method of making an immune receptor modifier conjugate of formula (I):

wherein R² is a biotic ligand, X¹ is OH or SH, R¹ is linear alkyl, branched alkyl, substituted alkyl, unsubstituted alkyl or alkoxyalkyl, X² is a coupling group; and m and n each are independently an integer selected from 1 to 10, said method comprising reacting a coupling precursor

with the biotic ligand R² to yield said immune receptor modifier conjugate.
 9. Use of the immune receptor modifier conjugate of claim 1 or salts thereof, for preparing therapeutic drugs at various ratios, for preparing compound drugs at various ratios with other drugs, or for preparing complexes or combinations with pharmaceutically acceptable carriers.
 10. Use of the immune receptor modifier conjugate of claim 1 for immunomodulation, antibody preparation, anti-virus, diabetes, tumor immunomodulation and tumor biological-immunotherapy.
 11. The immune receptor modifier conjugate of claim 2, characterized in that the conjugate comprises the compounds of formulae 1-1, 1-2, 1-3 and 2-1:


12. The immune receptor modifier conjugate of claim 2, characterized in that the conjugate comprises:

wherein the biotic ligand R² represents polypeptide or protein; MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate 5-3 5-4 5-5 5-6 5-7 5-8 5-9 5-10 5-11 Molecular 36326 3396 1272 88132 22233 40420 50724 36067 18259 weight Coupling 4 1 1 5 3 4 6 2 2 degree R² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 5-12 5-13 5-14 5-15 5-16 5-17 5-18 5-19 5-20 Molecular 86890 3357 1424 75029 194385 140469 55430 35834 33353 weight Coupling 6 1 1 5 3 6 3 6 4 degree


13. The immune receptor modifier conjugate of claim 2, characterized in that the conjugate comprises:

wherein the biotic ligand R² represents polypeptide or protein; MUC1 R² NANOG (epitope) MG7 POSTN Twist Anxa1 Akt1 CD47 Sp17 Conjugate 6-3 6-4 6-5 6-6 6-7 6-8 6-9 6-10 6-11 Molecular 36630 3469 2257 88512 22461 40724 51362 36219 18411 weight Coupling 4 1 1 5 3 4 6 2 2 degree R² PSMA M2e NP₃₆₆₋₃₇₄ SGLT2 PEAK1 HER2 MMP-10 PD-L1 PD-1 Conjugate 6-12 6-13 6-14 6-15 6-16 6-17 6-18 6-19 6-20 Molecular 87346 3433 3460 75410 194614 140925 55659 36290 33657 weight

wherein R² in conjugate 6-14 is NP₃₆₆₋₃₇₄ trimer, and R² in conjugate 6-5 is MG7 trimer. 